Study On The Interaction Of Quinoline And Isoquinoline Alkaloids Luminescent Probes With CtDNA

Chapter 1 In this chapter,following questions were elaborated:the composition of DNA and characteristics of its structure;binding modes of small molecules with DNA and fators affect their interactions.Furthermore,the theoretical research and present situations of some common techniques used in this field were reviewed.Chapter 2 The fluorescence and absorption spectra behaviors of palmatine in different solvents with different polarities and under different pH were studied.The results show that,the absorption intensity of palmatine increases and red shifts with the increase of the polarity of the solvents.The fluorescence intensity and spectra of palmatine are affected by the polarity of the solvents obviously as well.Only the second fluorescence emission peak blue shifts and increases with the decrease of the polarity of solvents.Effects of pH on the spectra of absorption and fluorescence are not great.The fluorescence quantum yields of palmatine in different solvents were measured.It can be drawn from this chapter that palmatine is sensitive to the surrounding environment.Chapter 3 The interaction of palmatine with DNA was studied mainly by absorption and fluorescence spectrometry.Many spectral parameters change after the addition of DNA, such as the intensity of fluorescence,absorbance,fluorescence quantum yield and polarization,etc.It is found that palmatine acts as a light switch,its fluorescence is very weak in the absence of DNA,but in the presence of DNA,its fluorescence is obviously enhanced.After the addition of DNA,the absorption spectra of palmatine red shift and its absorbance decreases,but its polarization and fluorescence quenching extent by KI do not increase.The binding constant was measured and the binding mode and mechanism were discussed.The results show that the binding mode is mix-mode,that is to say,intercalating, groove,and electrostatic binding are all exist.Among them,groove binding is a major factor affecting the binding ability.Binding constant measured is 2.52(±0.15)×10⁴L/mol, and binding site number is 0.16.Chapter 4 The PS-RTP spectra properties of palmatine were investigated.Experiments conditions were studied in detail.Palmatine can emit strong RTF on the slow speed filter paper directly.Using TIAc as perturber,it can emit strong room temperature phosphorescence.Its maximum excitation is 350nm,emission are 522nm and 615nm. When the concentration of heavy atom higher than 2mol/L and in pH 6-7 medium,its RTP intensity reaches to the maximum.PS-RTP analytical method was established under the optimum experimental conditions.At the same time,the interaction of palmatine with DNA was investigated by PS-RTP and PS-RTF.The effect of quencher to the luminescence intensity was studied,and the binding mechanism and binding mode were discussed.This chapter further demonstrates that the binding mode of palmatine with DNA is mixed mode.This is in accordance with the conclusion in chapter 3.Chapter 5 The PS-RTP and PS-RTF spectra properties of berberine and jiatrorrhizine were investigated.Experiment conditions were studied in detail.Berberine and jiatrorrhizine can emit strong RTF on the slow speed filter paper directly.Using TINO₃ as heavy atom perturbation,berberine can emit strong RTP.Its maximum excitation is 353nm, maximum emission are 520nm and 619nm.The RTP of jiatrorrhizine is not observed under the same condition.At the same time,the interactions of berberine and jiatrorrhizine with DNA was investigated by PS-RTP and PS-RTF techniques,absorption and fluorescence spectrometry were used as well.Binding constants were determined by absorption and fluorescence spectrometry,and the binding mechanism and mode were discussed. Intercalation was proved as the binding mode.The evaluation of binding constants shows that,the binding affinities of the two protoberberines with DNA are in the order of berberine＞jiatrorrhizine.Chapter 6:The interactions of chelerythrine and sangunarine with DNA were mainly studied by absorption,fluorescence spectrometry,fluorescence polarization,and denatured DNA methods.Their binding modes and binding affinities with ctDNA were researched. The results show that at pH5.40,the addition of DNA leads to the increase of the fluorescence intensity of chelerythfine,while the intensity of sangunarine is obviously quenched.The phenomena of hypochromicity and bathochromic shifts of their absorption spectra prove that their binding modes with DNA are both intercalation.Same result can be drawn from the fluorescence polarization experiments.The two alkaloids can intercalate into the double helix of DNA.Under some degree of ion strength,this affinity will become weak.Binding constants for chelerythrine and sangunarine with DNA are 2.98×10⁵L/mol and 8.86×10⁵ L/mol respectively,binding site are 0.210 and 0.18 respectively.In addition,electroanalytical method were studied,and it shows that there exists nonspecific electrostatic binding.Sangunarine shows stronger binding ability with DNA than chelerythrine.Chapter 7 The interaction of morphine with DNA was studied by absorption, fluorescence spectrometry,fluorescence polarization,and denatured DNA methods in neutral Tris-HCl buffer.The results show that morphine forms stable complex with DNA. The effects of concentration,pH and temperature on the interaction of morphine with DNA were investigated.The binding mode and binding mechanism were discussed. Experiments of ion strength,anion quencher,fluorescence polarization and denatured DNA all indicate that the binding mode is intercalation.With the increase of temperature, the quenching extent of DNA to morphine becomes weak.This proves that the fluorescence quenching of morphine is in accordance with static mechanism.In addition, the binding constant and binding site were measured.The results show that the binding affinity is not very big and morphine can form 1:1 complex with DNA.Chapter 8 The fluorescence spectra behaviors of QN were researched.Selecting QN as luminescence probe,the interaction of QN with DNA was studied by means of absorption, fluorescence spectroscopy and fluorescence polarization.Factors effected the interaction was studied,and the mechanism and binding mode were discussed.It is found that the fluorescence intensity is quenched greatly,and the fluorescence polarization increases.QN can binds to DNA by intercalation mode.Chapter 9 This chapter summarized all the experimental results in this thesis.Further works were proposed as well.